Intraspecific trait plasticity to N and P of the wetland invader, Alternanthera philoxeroides under flooded conditions

Abstract Interactions between invaders and resource availability may explain variation in their success or management efficacy. For widespread invaders, regional variation in plant response to nutrients can reflect phenotypic plasticity of the invader, genetic structure of invading populations, or a combination of the two. The wetland weed Alternanthera philoxeroides (alligatorweed) is established throughout the southeastern United States and California and has high genetic diversity despite primarily spreading clonally. Despite its history in the United States, the role of genetic variation for invasion and management success is only now being uncovered. To better understand how nutrients and genotype may influence A. philoxeroides invasion, we measured the response of plants from 26 A. philoxeroides populations (three cp haplotypes) to combinations of nitrogen (4 or 200 mg/L N) and phosphorus (0.4 or 40 mg/L P). We measured productivity (biomass accumulation and allocation), plant architecture (stem diameter and thickness, branching intensity), and foliar traits (toughness, dry matter content, percent N, and percent P). A short‐term developmental assay was also conducted by feeding a subset of plants from the nutrient experiment to the biological control agent Agasicles hygrophila, to determine whether increased availability of N or P to its host influenced agent performance, as has been previously suggested. Alternanthera philoxeroides haplotype Ap1 was more plastic than other haplotypes in response to nutrient amendments, producing more than double the biomass from low to high N and 50%–68% higher shoot: root ratio than other haplotypes in the high N treatment. Alternanthera philoxeroides haplotypes differed in seven of 10 variables in response to increased N. We found no differences in short‐term A. hygrophila development between haplotypes but mass was 23% greater in high than low N treatments. This study is the first to explore the interplay between nutrient availability, genetic variation, and phenotypic plasticity in invasive characteristics of the global invader, A. philoxeroides.


| INTRODUC TI ON
Invasive species are recognized as major economic and ecological hazards, with the frequency and magnitude of their negative impacts expected to increase in the future (Diagne et al., 2021).
Determining the influence of local site conditions on invader establishment, spread, and abundance is of paramount importance for prioritizing areas for management and predicting impacts associated with invasions (e.g., Godoy et al., 2011;Kolb & Alpert, 2003;Woo & Zedler, 2002). Studies that examined invader response to competition or nutrient enrichment have advanced our understanding of traits associated with successful invaders and provided research directions to develop effective conservation plans, with the goal of reducing the likelihood of impacts and increasing species diversity overall (Buckley, 2008;Larson et al., 2011).
Within aquatic and wetland ecosystems, increased availability of nitrogen and phosphorus (i.e., eutrophication) has emerged as a key variable in a number of successful invasions and has attracted research focus to mitigate its prevalence globally (Carson et al., 2018;Gérard et al., 2014;Scherer-Lorenzen et al., 2008). The role of nutrient enrichment in plant invasions has been examined for a number of systems (e.g., Fan et al., 2013;Yu et al., 2018) and in addition to increasing the likelihood of negative impacts caused by the invader, elevated nutrient levels can impact when and how management is applied (Elgersma et al., 2017;Room et al., 1989). For example, successful biological control of weeds with insects can be highly dependent on plant nutrition, with significant effects of nitrogen on the timing of agent establishment (Room & Thomas, 1985), population buildup (Harms & Cronin, 2019;, and dispersal (Wilson et al., 2007). The relationship between eutrophication and herbicide efficacy is less clear, but there are numerous examples of increased herbicide efficacy when plants are grown with supplemental fertilization before application (Aulakh, 2020;Cathcart et al., 2004). Invader response to fertilization or management may depend on genetic structure of invasions, resulting from sexual recombination or the introduction and establishment of populations from multiple native range source locations (Ward et al., 2008). The ways in which genetic variation and eutrophication interact during biological invasions is largely unstudied but has begun to receive interest (Harms, Cronin, & Gaskin, 2021;Holdredge et al., 2010;Kettenring et al., 2011;Liao et al., 2016), especially with ever-increasing nutrient inputs to natural systems (Hale et al., 2015). For example, the aquatic invasive plant flowering rush (Butomus umbellatus L.) has multiple genotypes and at least two cytotypes in the United States (Kliber et al., 2005), and previous work demonstrated that diploid and triploid populations differed significantly in their growth, resource allocation, and chemical response to N or P (Harms, Cronin, & Gaskin, 2021). In the southeastern United States, the aquatic plant Alternanthera philoxeroides (Mart.) Griseb. has been introduced at least twice (Kay & Haller, 1982), with introduced biotypes displaying differential susceptibility to herbicides (Kay, 1992) and to biological control agents (Pan et al., 2012). Despite considerable interest in managing the spread and establishment of A. philoxeroides, the possible interaction between genetic variation within invading populations and nutrient response and allocation of plants is unknown.
To better understand how genetic variation and nutrient availability interact to promote invasions and their management, we conducted a common garden experiment with the aquatic invader, A. philoxeroides. We grew plants under combinations of low or high nitrogen and phosphorus, then measured (1) key growth, structural, and elemental chemistry traits, and (2) short-term performance of biocontrol agents fed leaves of plants grown under different nutrient combinations. Based on previous studies, we expected the broad-stemmed morphotype (Ap1 sensu Williams et al., 2020) to display the greatest growth response to increased nutrient levels.
We additionally expected elevated nutrients to translate into higher tissue nutrients and faster development of the alligatorweed flea beetle. This is the first study to link genetic variation in a widespread clonal invader, invader performance and phenotype, and management with biological control.

| Study system
Alternanthera philoxeroides is an aquatic macrophyte native to South America, which has been introduced in over 32 countries around the world (Tanveer et al., 2018). Alternanthera philoxeroides is a common and widespread invader in the Southeastern United States but there are disjunct populations in California (Walden et al., 2019).
There are at least six haplotypes of alligatorweed in the United States, likely from multiple introductions (Williams et al., 2020) (Buckingham, 1996). The most common agent in the United States is A. hygrophila but low winter temperatures limit its distribution to warm southern portions of the range of A. philoxeroides .
The introduction history of A. philoxeroides into the United States is unclear. Alternanthera philoxeroides is thought to have been first introduced into the country via ship ballast water release and was

T A X O N O M Y C L A S S I F I C A T I O N
Applied Ecology, Biogeography, Ecophysiology, Evolutionary Ecology, Invasion Ecology recorded in Alabama and Louisiana prior to 1900 (Zeiger, 1967).
However, there are no subsequent records of introductions since then, and any introductions that may have occurred would have been cryptic. As early as 1982, it was clear that multiple genotypes of A. philoxeroides were present, with differences in traits between them (Kay & Haller, 1982). It has been further acknowledged that multiple introductions likely occurred but, until recently, the degree of genetic diversity and its role in invasive plant management in the United States was unknown (Geng et al., 2016;Williams et al., 2020).

| Plant culture
Alligatorweed clones were collected from 26 locations across the entire US distribution ( Table 1)  Hoagland's nutrient solution (Hoagland & Arnon, 1950). During the growing season and leading up to experiments, nutrients were exchanged biweekly. At other times, nutrients were exchanged monthly. Plants were periodically pruned and thinned to maintain low culture biomass when not needed, and pests (e.g., whiteflies, prob. Trialeurodes sp.; aphids, Myzus persicae) were treated with insecticidal soap as they were observed. Pest control continued throughout the year but ceased within 4 weeks of experiments, and fresh pest-free cuttings were used to start experiments.

| Haplotyping
Alligatorweed populations used for this experiment were the same as reported by Williams et al. (2020). To determine haplotype, three apical meristems were collected from each culture, dried in silica gel, then DNA was extracted from all samples (n = 78) using the IBI Scientific MINI Genomic DNA kit (Plants) (Dubuque, Iowa) as per the manufacture's instructions. Plants were genotyped using primers developed around mononucleotide repeats in three chloroplast (cpDNA) regions (rpL16, trnS-G, trnF intron/trnL-F spacer) using previously described methods (Williams et al., 2020)

| Experimental setup
To test haplotype response to combinations of nitrogen (N) and phosphorus (P), we conducted a three-way factorial randomized block design experiment with three replicates of each nutrient treat- Temperature in the greenhouse was maintained between 15 and 25°C, and we used 60% shade cloth on the greenhouse for the duration of the experiment.
Four nutrient solutions were prepared, each containing a combination of low or high nitrogen or phosphorus (Table 2) The experiment was harvested after 5 weeks, at which time measurements were taken. To measure leaf toughness, two leaves per plant were randomly chosen, placed on a round PVC surface with a 10-mm hole, then a force gauge was used to measure the force (in pounds) required to push a 9-mm-diameter cylindrical rod through the leaf. Length of the main stem, the number of branches on the main stem, and sum total length of branches per stem were measured. Because A. philoxeroides produces hollow stems under flooded conditions, and stems are the pupation site of A. hygrophila (Maddox et al., 1971), stem diameter and stem wall thickness were measured at the thickest point of each stem using a digital caliper.
Two leaves from each plant were collected separately, weighed, and then used in the A. hygrophila development assay described in the next section. The remaining leaves were separated from stems, weighed, and placed in paper bags for drying. Dry weights of the leaves removed for the A. hygrophila feeding assay were estimated from the dry-to-wet weight ratio of the remaining leaves at harvest, then added to aboveground biomass measurements for each plant.
Similarly, stems and roots (belowground plant parts) were separated and rinsed with reverse osmosis water, then placed in paper bags for

| Larval development assay
In addition to plant measurements described above, we evaluated the role of nutrients in A. philoxeroides herbivore resistance with a short-term feeding assay. Prior to biomass harvest in the above experiment, two leaves were excised from each plant (n = 720 leaves,

| Statistical approach
To examine whether there were genetic-based differences in nutrient responses between alligatorweed haplotypes (Ap1, Ap3, Ap6), we used the Akaike information criterion adjusted for small sample size (AICc) to select the most informative mixed model (Proc MIXED; SAS 9.3, SAS Institute, Cary, NC) from the full set of candidate models (Burnham & Anderson, 2003). Candidate models were constructed from the simplified full model (haplotype, nitrogen level, phosphorus level, and all two-way interactions) with the constraint that an interaction term was included only if their main effects were also included in the model. In all models, A. philoxeroides population (nested within haplotype) was included as a random variable, location within the greenhouse was a blocking factor, and initial fresh weight of propagules was included as a covariate. Also, because latitudinal clines in growth, reproduction, or herbivore defense traits The top model had the lowest AIC of all candidate models, and ΔAICc was calculated as the difference between the top model and all others. Models with AICc <2 were considered to have substantial support (Burnham & Anderson, 2003). Akaike weights are also reported, which represent the relative likelihood that the model is the best given the data and other candidate models (Burnham & Anderson, 2003 We then applied the J correction for small sample size using a sample size of six per treatment combination (Borenstein et al., 2011):

| Response of A. philoxeroides haplotypes to elevated nutrients
In nearly all measured response variables, plant haplotype was a significant explanatory variable based on AICc model selection ( Table 3).
Alternanthera philoxeroides haplotype, both nutrients, latitude, and their interactions were influential in explaining biomass production and allocation. Although four candidate models received support (ΔAICc ≤2) for dry weight (DW) biomass, the top model included only haplotype, nitrogen, phosphorus, and the haplotype × nitrogen interaction (R 2 (m) = 0.80; R 2 (c) = 0.82). Total biomass for all three haplotypes was similar at 2 mg/L N, but 71% and 105% greater for Ap1 at 200 mg/L than Ap3 and Ap6 haplotypes, respectively (Figure 1a).
Branching intensity was influenced by the interaction between N and P and increased 22% from low to high P at low levels of N and 78% for high N treatments (Figure 2a). Branching intensity was also higher for Ap3 and Ap6 haplotypes at low N only (Figure 2b).
Variation in stem diameter, stem wall thickness, and leaf toughness variables were all explained by haplotype, nitrogen, and the haplotype х nitrogen interaction ( Table 1). Stem diameter was consistently higher for haplotype Ap1 and increased 92% from low to high levels of N, whereas Ap3 and Ap6 stem diameter increased 64% and 33% over the same N treatments (Figure 3a). Stem wall thickness followed a similar pattern-stem walls of haplotype Ap1 plants were consistently thicker than other haplotypes and increased with N 29% (Ap1), 24% (Ap3), and 14% (Ap6) (Figure 3b). Alternanthera philoxeroides leaf toughness decreased with increasing N, although the decrease was greatest for Ap1 plants (Figure 3c). Dry matter content was not influenced by treatment variables in the experiment and was best explained by the interceptonly model (AICc = −1386.8; AICc weight = 1.0).

TA B L E 3
Top best-fit models for each dependent variable, based on AICc selection procedure.  Percent leaf phosphorus increased with phosphorus treatments, but the magnitude of increase was highest in high nitrogen treatments, regardless of haplotype (Figure 4c; 123% increase from low to high phosphorus).

F I G U R E 1
Influence of nutrients on biomass production (g DW; least squares mean ± SE) (a) and allocation (b, c) of A. philoxeroides haplotypes. These interactions were identified as influential based on model selection. Error bars obscured due to small size.

F I G U R E 2
Influence of the nitrogen × phosphorus interaction (a) and the haplotype × nitrogen interaction (b) on branching intensity (least squares mean ± SE). These interactions were identified as influential based on model selection. Error bars obscured due to small size.

F I G U R E 3
Influence of the haplotype х nitrogen interaction on A. philoxeroides stem diameter (a), stem wall thickness (b), and leaf toughness (least squares mean ± SE) (c). These interactions were identified as influential based on model selection. Error bars obscured due to small size.
In the short-term feeding assay, Agasicles hygrophila weight was influenced by the nitrogen × phosphorus interaction (Table 1; AICc = 562.6; AICc weight = 0.35). Regardless of A. philoxeroides haplotype, larval weight was greater overall in the high nitrogen treatment, increasing 23% from low to high phosphorus ( Figure 5).
Ap1 plants displayed two times the plasticity for leaf toughness than Ap3 plants and three times that of Ap6 plants (Ap1 g = −0.67 ± 0.08, Ap3 g = −0.303 ± 0.12, Ap6 g = −0.19 ± 0.08) but only 7% more plasticity than Ap3 and 27% more than Ap6 in percent leaf nitrogen in response to nitrogen treatments. Response of percent leaf phosphorus, DMC, and resistance to herbivory (A. hygrophila weight) were similar between haplotypes in response to nitrogen. In contrast to the strong response to nitrogen, we detected no differences between haplotypes in plasticity of any variables to phosphorus treatments ( Figure 6).

| DISCUSS ION
For invaders with multiple introduced genotypes, determining how environmental variation contributes to their success, and whether the response to the environment varies by genotype, can improve predictions about which invaders (or genotypes) will become invasive (i.e., using a trait-based approach; Bhattarai et al., 2017;Davidson et al., 2011;Weinig et al., 2007) and guide decision-making around management actions (Gaskin et al., 2011;Thum, 2018).
There are a growing number of invaders for which it is clear that management should be tailored at the subspecific level (Blossey & Casagrande, 2016;Croy et al., 2020;Harms, Shearer, et al., 2020;Harms, Williams, & Purcell, 2021), and this work further supports continuation of that research direction. How genotypes of introduced species differ in their tolerance or response to environmental variation is key to distinguishing invasive traits, and thus modeling their distribution and impacts in the invaded range.
Our results demonstrate that there is significant variation in A. philoxeroides haplotype-specific responses to elevated nutrients.
We did not directly test whether increased plasticity is adaptive for F I G U R E 4 Influence of nitrogen × phosphorus interaction (a), haplotype × nitrogen interaction (b) on A. philoxeroides leaf nitrogen and nitrogen × phosphorus interaction on leaf phosphorus (c) (least squares mean ± SE). These interactions were identified as influential based on model selection. Error bars obscured due to small size.

F I G U R E 5
Influence of the nitrogen × phosphorus interaction on resistance to herbivory (A. hygrophila larval weight; least squares mean ± SE of untransformed data). The interaction was identified as influential based on model selection.
A. philoxeroides; however, for a plant that reproduces primarily by clonal fragmentation, we provided evidence for increased fitness through several proxies, including variation in biomass production and branching intensity related to haplotype and nutrient level. Increased branching, for example, may lead to increased matting on the water surface and more propagules available for dispersal upon disturbance. This is one of only a few studies to examine intraspecific variation in plant traits of A. philoxeroides in the United States, and joins a growing number of studies in the invasion biology literature that demonstrate (1) intraspecific variation in response to biotic or abiotic environmental variables within an invading species (Harms, Cronin, & Gaskin, 2021;Lavergne & Molofsky, 2007), and (2) the potential importance of phenotypic plasticity for invader success (Bhattarai et al., 2017;Castillo et al., 2014;Chevin & Lande, 2011;Eller & Brix, 2012;Geng et al., 2016).

| Nutrients and management
In the United States, A. philoxeroides has a large geographic distribution that includes the entire southeastern region and California.
Of the six recognized haplotypes in the United States, Ap1 has the broadest latitudinal distribution, collected from southern Florida and Texas north to Arkansas and North Carolina (Williams et al., 2020). Alternanthera philoxeroides haplotype Ap1 had a similar biomass response at low nutrients as Ap3 and Ap6 but the greatest response to elevated nutrients by most measures, which suggests that it is well-adapted to a range of nutrient conditions. The increased response of invasive weeds over native species to high resource availability can exacerbate the displacement of native species (e.g., Leishman & Thomson, 2005;Richardson & Pyšek, 2006;Rickey & Anderson, 2004), which may explain the broad F I G U R E 6 Plasticity (g; mean ± 95% CI) of A. philoxeroides haplotypes in response to nitrogen (black circle) or phosphorus (white circle) treatments.
geographic distribution of Ap1. Also, although there is substantial overlap in the areas where A. philoxeroides haplotypes occur, there is often only a single haplotype within a site (Williams et al., 2020).
Whether there is some spatial segregation based on nutrient conditions is unknown, but the differences in haplotype response to nutrients may explain some of that variation. Ap1 had the highest biomass response to elevated nutrients, largely in the form of shoot production and branching, suggesting that a competitive advantage over other species may stem from shading in addition to nutrient use. In contrast, Ap6 plants grew slower but allocated a larger proportion of biomass to roots overall and produced more biomass and higher branching in low nutrient treatments, which may be advantageous and explain marginally greater assimilation of N under low nutrient conditions (Figure 4b).
In addition, the current study used only the three most common A. philoxeroides chloroplast haplotypes to evaluate variation in plasticity, but there are a number of other rare ones in the invaded range. For example, California populations so far consist of only Ap2 and Ap4 haplotypes, which are only known to occur in one other state each (Ap2 in Georgia, Ap4 in Arkansas) (Williams et al., 2020). Ap5 was only detected in a single location in Louisiana and may not occur anywhere else in the United States. Investigation into other, rare haplotypes may provide important insights into their ecology and potential to become more widespread and invasive.
We did not find differences in herbivore resistance between A. philoxeroides haplotypes, but the influence of plant nutrition was clear. Differences in A. hygrophila development time on narrow and broad-leafed A. philoxeroides have been demonstrated (Pan et al., 2012(Pan et al., , 2013 and had we allowed development to continue in our experiment, we may have observed similar differences between haplotypes Ap1 and Ap6, which are the morphological analogs to narrow and broad-leafed A. philoxeroides (Williams et al., 2020). However, prolonging the assay would have increased the volume of biomass removed from the treatments, delaying collection, and potentially altering the final plant measurements. Thus, the window for development was intentionally kept short.
Despite the short development time, we found the influence of elevated nutrients on several plant traits that influence successful biological control. For example, at elevated nitrogen treatments, foliar nitrogen increased, DMC decreased, and leaf toughness decreased-all results which should improve the development of the foliar-feeding A. hygrophila. In fact, previous work demonstrated that, at high nitrogen levels, larval development time decreased and larval survival increased (Harms & Cronin, 2019). Although larval development would be expected to accelerate on all haplotypes, Ap1 is most likely to have the largest influence on development time, if development was allowed to continue through eclosion. Stem diameter was largest overall for Ap1 plants, and also responded most to increased nitrogen. However, stem wall thickness also increased in response to nitrogen, which may have a confounding impact on larval A. hygrophila, if given sufficient time to develop and pupate.
The diameter of A. philoxeroides stems is important for A. hygrophila pupation, a feature which has been suggested to possibly limit persistence of agents in some populations, particularly those growing in terrestrial habitats (Ma & Wang, 2004;Pan et al., 2011).
Herbicide control of A. philoxeroides can be highly effective, especially in areas where biological control does not provide adequate control or where agents have not been introduced (Clements et al., 2017). Although herbicide tools are available, which to use and where to use them largely depends on adjacent land-use and whether the plants are growing in flooded or dry habitat (Dugdale & Champion, 2012). To our knowledge, the roles of plant genotype and nutrient availability on herbicide efficacy have not been addressed for any emergent or floating weeds in the United States, though evidence for variation in herbicide susceptibility among genotypes of some invaders is mounting (e.g., Chorak & Thum, 2020). Two variables that may influence herbicide effectiveness in A. philoxeroides are branching architecture and biomass allocation. The greater the proportion of biomass allocated to roots, the more difficult it is to achieve adequate multiyear control of A. philoxeroides (Clements et al., 2017;Schooler et al., 2008). Ap6 plants had the lowest shoot: root ratio overall, regardless of nutrient treatment, and the ratio increased the least from low to high nitrogen or phosphorus. This finding, and that the highest biomass overall and greatest biomass response to increased nutrients occurred for Ap1, suggests that Ap6 plants may be least affected by herbicide application, all other variables held constant. Additionally, increased proportional allocation to aboveground growth under high nutrient conditions may make Ap1 more susceptible to biocontrol and herbicides targeting emergent growth.
It has been suggested that highly plastic species are the most successful invaders because they can tolerate a wide variety of biotic and abiotic conditions (Davidson et al., 2011;Higgins & Richardson, 2014;Richards et al., 2005). Outstanding questions remain about whether species or genotypes have the observed level of plasticity upon introduction, or whether they evolve increased plasticity in novel habitats. The widespread success of A. philoxeroides is thought to be largely a result of plasticity to environmental variation (e.g., to water availability; Geng et al., 2016), a pattern that has not differed between native and introduced ranges in earlier studies, and suggests that A. philoxeroides did not evolve greater plasticity during invasion in the United States. However, previous studies did not test a genetically diverse subset of populations from the invaded-range, or make explicit comparisons between introduced genotypes to evaluate the range of plasticity within the invasion. In the current study, we demonstrated high plasticity to nitrogen, but the degree of plasticity varied among measured traits and haplotypes.
Differences in response to elevated levels of nitrogen and phosphorus were observed among common A. philoxeroides haplotypes found within their invasive range. It is unsurprising that the haplotype with the greatest degree of plasticity (Ap1) in traits affecting spread and management is also the most prevalent. Although there is limited evidence for differences in efficacy of herbicide and biological control among A. philoxeroides haplotypes, these differences have yet to be explicitly explored. There has also not been any investigation into how this plasticity affect intraspecific competition among haplotypes or influences their geographic distributions.
More work is needed to fully evaluate conditions that influence A. philoxeroides invasion success, and field verification of haplotype distribution in relation to temperature or nutrient responses would improve predictions about locations at risk for future establishment and spread.

ACK N OWLED G M ENTS
This research was conducted under the work unit, "Improving

FU N D I N G I N FO R M ATI O N
No external funding was provided for this work.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.